Bottom Line:
Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers.It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot.In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

ABSTRACTFormylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

Mentions:
To gauge the stability profile of the StPurL protein thermal denaturation was investigated by following the unfolding of secondary structure elements, more specifically unfolding of α helices, with increasing temperature. The protein appeared to follow a three state unfolding mechanism in two seemingly cooperative steps (Figure 2A). The first unfolding transition at 42°C led to a loss of 15–20% of the secondary structure content of the protein and 80% loss in activity (Figure 2B). The second unfolding transition at 80°C represented unfolding of the bulk of the protein. Domains in a protein may unfold independently or cooperatively and unfolding of multidomain proteins presents few possible scenarios [5]. B-factors of the StPurL crystal structure (PDB code 1T3T) were analyzed to estimate the dynamic nature of various parts of the structure. It was observed that the N-terminal domain had the highest B-factors, followed by the linker domain whereas the glutaminase and the FGAM synthetase domains had lower B-factors (Figure 2C). Moreover, per-residue rmsd calculations between xenon bound and native structures showed a higher degree of variation in the N-terminal region. The above evidence suggests that the N-terminal and the flexible linker domains constituting 15–20% of the secondary structure (Figure 2D), unfold in the first transition at 42°C. Additionally, it has been shown previously that homologs of this protein cannot function efficiently in the absence of PurS protein (analogous to the N-terminal domain) [16] hence, 80% loss of activity upon its unfolding is in accordance with this previous observation. Evolutionary conservation scores calculated using the multiple sequence alignment used for SCA also corroborated this evidence, as the glutaminase and the FGAM synthetase domains exhibited high conservation scores in contrast to the N-terminal domains (Figure 2E). Based on this information, we have proposed a two-step unfolding mechanism for the StPurL protein which is depicted in Figure 2F.

Mentions:
To gauge the stability profile of the StPurL protein thermal denaturation was investigated by following the unfolding of secondary structure elements, more specifically unfolding of α helices, with increasing temperature. The protein appeared to follow a three state unfolding mechanism in two seemingly cooperative steps (Figure 2A). The first unfolding transition at 42°C led to a loss of 15–20% of the secondary structure content of the protein and 80% loss in activity (Figure 2B). The second unfolding transition at 80°C represented unfolding of the bulk of the protein. Domains in a protein may unfold independently or cooperatively and unfolding of multidomain proteins presents few possible scenarios [5]. B-factors of the StPurL crystal structure (PDB code 1T3T) were analyzed to estimate the dynamic nature of various parts of the structure. It was observed that the N-terminal domain had the highest B-factors, followed by the linker domain whereas the glutaminase and the FGAM synthetase domains had lower B-factors (Figure 2C). Moreover, per-residue rmsd calculations between xenon bound and native structures showed a higher degree of variation in the N-terminal region. The above evidence suggests that the N-terminal and the flexible linker domains constituting 15–20% of the secondary structure (Figure 2D), unfold in the first transition at 42°C. Additionally, it has been shown previously that homologs of this protein cannot function efficiently in the absence of PurS protein (analogous to the N-terminal domain) [16] hence, 80% loss of activity upon its unfolding is in accordance with this previous observation. Evolutionary conservation scores calculated using the multiple sequence alignment used for SCA also corroborated this evidence, as the glutaminase and the FGAM synthetase domains exhibited high conservation scores in contrast to the N-terminal domains (Figure 2E). Based on this information, we have proposed a two-step unfolding mechanism for the StPurL protein which is depicted in Figure 2F.

Bottom Line:
Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers.It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot.In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

ABSTRACTFormylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.